ABSTRACT Sub-acute lung infections are increasingly recognized as drivers of poor symptom control among a subset of individuals with chronic lung disease (estimated more than 2 million for Asthma and COPD patients). When these sub-acute infections are diagnosed and treated appropriately, chronic lung disease patients can convert from moderate/severe to a milder disease phenotype, requiring lower medication to achieve better health at a significantly lower cost. Current gold-standard diagnostics for sub-acute infection rely on decades-old technology that can take weeks to complete, have limited sensitivity, and are limited in the type and number of microbes that can be screened by a single test. Thus, a critical gap exists due to the inability of current diagnostics to comprehensively, quickly, and accurately detect microbial pathogens in low-burden clinical samples, which is a significant barrier to improved clinical outcomes in chronic lung disease. We developed a comprehensive NGS targeted amplicon panel for a) detection and profiling of microbes with associated treatment resistance data and b) profiling human immune system host response genes. Our NGS diagnostics (Dx) panel is a significant technological innovation over current methodology due to the combination of the inherent technical characteristics of a targeted NGS approach, the specific set of amplicons we target and our proprietary laboratory and analysis workflows. The long-term goal of this project is to provide a novel clinical tool for the detection of low-burden microbial infections driving disease pathology, symptomology, and exacerbations in chronic lung disease populations such as COPD, cystic fibrosis, and moderate to severe Asthma. Our preliminary studies demonstrate an ability to a) utilize patient samples directly vs. requiring cultures, b) provide orders of magnitude greater sensitivity and accuracy than qPCR or metagenomic approaches, and c) comprehensively screen bacterial, fungal and viral species in a single assay. Our specific aims are designed to test the feasibility of our NGS Dx approach to provide substantial enhancements in accuracy, sensitivity, specificity, and speed over current clinical standards. The first aim of this study is to establish the limits of detection and specificity of the targeted NGS Dx panel to detect microbial species when most of the DNA/RNA in the sample comes from a human host. The second aim of the study is to demonstrate the feasibility of the NGS Dx panel to identify sub-acute infections among subjects with severe chronic lung disease. After successful completion of these aims, Phase II studies will build off of these and other early research activities to demonstrate clinical validation and utility in a CLIA certified laboratory, enabling engagement of patient samples for clinical diagnostic testing. The total market for this diagnostic is the set of chronic lung disease patients with uncontrolled symptoms who could be screened for sub-acute infections. Our competitive advantages include vastly improved sensitivity, speed, comprehensive microbe detection, microbe profiling, stream-lined analysis, and treatment effectiveness insights within a single assay.